The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, colony stimulating factors and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides xe2x80x9cdirectlyxe2x80x9d in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent xe2x80x9cindirectxe2x80x9d cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed.
Meningiomas are brain tumors formed from cells of the meninges, which are membranes that cover the brain and spinal cord. Meningiomas are relatively common and account for roughly half of all primary tumors of the brain and spinal cord. They are generally benign and slow growing, but may cause serious neurological problems due to invasion of or pressure on surrounding brain tissue. Treatment options include surgical removal and radiation therapy.
Astrocytomas are brain tumors formed from astrocytes, a type of brain glial cell that provides physical and nutritional support to the neurons of the brain. Astrocytomas are also a common tumor of brain tissue origin and may vary in aggressiveness, from the very aggressive glioblastoma multiforme, to the moderately aggressive anaplastic astrocytoma, to the least aggressive astrocytoma. They spread by infiltrating surrounding brain tissue but usually do not metastasize to other parts of the body. Treatment options include surgical removal, radiation therapy and chemotherapy, but complete surgical removal is typically difficult if not impossible due to the extensive infiltration of normal tissue.
Breast cancer is one of the most common of all malignancies. In the United States, the cumulative lifetime probability of developing breast cancer is 12% and of dying from breast cancer is 3.5%. Staging and prognosis are usually based on invasion of lymph nodes; each additional positive lymph node is associated with a worse prognosis. In late stages of the disease, the breast cancer has metastasized to distant organs. More than 80% of breast cancers are of the invasive ductal type. The next most common variety, infiltrating lobular, constitutes almost 10% of all breast cancers. Medullary carcinoma represents about 5% of all breast cancers and is less likely to metastasize to regional lymph modes. The remaining 5% of breast cancers are generally less malignant. Treatment usually consists of surgical removal followed by radiation therapy and/or chemotherapy.
Cancer of the prostate is the most common malignancy in men in the U.S. and is the second most common cause of cancer death in men older than age 55 (after carcinomas of the lung and colon). Some carcinomas of the prostate are slow growing and may persist for long periods without significant symptoms, whereas others behave aggressively. Over 95% of prostatic cancers are adenocarcinomas that arise in the prostatic acini. The remaining prostatic cancers are divided among squamous cell and transitional cell carcinomas that arise in the ducts, carcinoma of the utricle, carcinosarcomas that arise in the mesenchymal elements of the gland, and occasional metastatic tumors. Treatment typically involves surgery, radiation therapy, and/or anti-androgen therapy.
Colon cancers are also a very common malignancy and typically are adenocarcinomas, or sometimes carcinoid tumors. Treatment is primarily surgical resection of the colon, although chemotherapy has been found to be beneficial in some cases.
Melanoma is a skin cancer which originates from melanocytes present in the epidermis and dermis. This cancer affects approximately 32,000 individuals per year in the United States. The incidence of melanoma has dramatically increased over the past 40 years. There are four types of melanoma. Three types, superficial spreading melanoma, lentigo maligna melanoma and acral lentigious melanoma have a period of superficial growth and the tumor does not penetrate deeply. These superficial tumors can be treated by surgical excision. The fourth type of melanoma, nodular melanoma, has a radial growth phase and is usually a deep invasive lesion which is capable of metastasis to any organ. These tumors can be treated with regional nodal dissection which may be complemented with chemotherapy, immunotherapy, chemoimmunotherapy and/or radiation therapy.
The types of sarcomas include osteosarcomas, fibrosarcomas, chondrosarcomas and Ewing""s tumor. Osteosacromas originate from osteoprogenitor cells. These tumors have a wide range of histopathology with at least 12 subtypes and may metastasize generally to the lung. These tumors are treated with amputation, wide resection, chemotherapy or radiation.
Lymphomas are neoplastic transformations of cells residing within lymphoid tissues. Non-Hodgkin""s lymphoma is derived primarily from B cells and is the most common neoplasm of patients between the ages of 20 and 40 years of age. There about 40,000 new cases of non-Hodgkin""s lymphoma each year in the United States and incidence is increasing with the incidence of AIDS. There is only a 30-40% rate of curability of non-Hodgkin""s Lymphoma. Conversely, it is unresolved which type of lymphoid cell from which Hodgkin""s disease derives. Hodgkin""s disease is a lymphoma which presents as a localized tumor that may spread to the contiguous lymphoid structures and eventually to other organs. Hodgkin""s disease is most prevalent in males between the ages of 15-20 years and then after the age of 50 years. There is a greater than 75% rate of curability of Hodgkin""s disease. Both non-Hodgkin""s lymphoma and Hodgkin""s disease are treated with radiotherapy, chemotherapy and salvage chemotherapy. In addition, non-Hodgkin""s lymphoma may be treated with bone marrow transplants.
Treatment options for cancer are of unpredictable and sometimes limited value, and there continues to exist a need for novel therapies and diagnostic methods for cancer conditions.
The compositions of the present invention include novel isolated polypeptides, in particular, novel EGF-repeat-containing polypeptides, isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, and antibodies that specifically recognize one or more epitopes present on such polypeptides. The novel EGF-motif-containing polypeptide is denoted herein as EGFL6. In prior applications this same polypeptide has been referred to as ERHyl.
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NOS: 3, 6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 1-502 of SEQ ID: 4 (The first amino acid residue in the sequence is designated as 1); a polynucleotide encoding a polypeptide comprising amino acid residues 1-21 of SEQ ID NOS: 6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 80-93 of SEQ ID NO:6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 95-128 of SEQ ID NO:6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 133-168 of SEQ ID NO:6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 175-214 of SEQ ID NO:6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 220-259 of SEQ ID NO:6 or 24; a polynucleotide encoding a polypeptide comprising amino acid residues 446-465 of SEQ ID NO:6 or 24; or a polynucleotide encoding a polypeptide comprising amino acid residues 363-365 of SEQ ID NO:6 or 24.
The isolated polynucleotides of the invention further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of SEQ ID NOS: 1, 2, 5 or 23; a polynucleotide comprising nucleotides 205-267 of the nucleotide sequence of SEQ ID NOS: 5 or 23 (The first nucleic acid residue of the sequence is designated as 1); a polynucleotide comprising nucleotides 442-483 of the nucleotide sequence of SEQ ID NOS: 5 or 23; a polynucleotide comprising nucleotides 487-588 of the nucleotide sequence of SEQ ID NOS: 5 or 23; a polynucleotide comprising nucleotides 601-708 of the nucleotide sequence of SEQ ID NOS: 5 or 23; a polynucleotide comprising nucleotides 727-846 of the nucleotide sequence of SEQ ID NOS: 5 or 23; a polynucleotide comprising nucleotides 862-981 of the nucleotide sequence of SEQ ID NOS: 5 or 23; a polynucleotide comprising nucleotides 1540-1599 of the nucleotide sequence of SEQ ID NOS: 5 or 23; a polynucleotide comprising nucleotides 1729-1731 of the nucleotide sequence of SEQ ID NOS: 5 or 23; or a polynucleotide comprising nucleotides 1291-1299 of the nucleotide sequence of SEQ ID NO:5 or 23.
The polynucleotides of the present invention still further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of a cDNA insert of clone pEGFR-HY1 deposited with the American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, Va., 20110-2209, U.S.A.); a polynucleotide comprising a nucleotide sequence of the cDNA insert of clone pEGFR-HY2 deposited with the ATCC; a polynucleotide comprising a nucleotide sequence of the cDNA insert of clone pEGFR-HY3 deposited with the ATCC; a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGFR-HY1; a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGFR-HY2; a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGFR-HY3; a polynucleotide comprising the full length protein coding sequence of SEQ ID NOS: 6 or 24 which polynucleotide comprises the cDNA insert of clone pEGFR-HY2, nucleic acids 323-357 of SEQ ID NOS: 5 or 23 and the cDNA insert of clone pEGFR-HY1; a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NOS: 6 or 24 comprising the cDNA insert of clone pEGFR-HY2, nucleic acids 323-357 of SEQ ID NOS: 5 or 23 and the cDNA insert of clone pEGFR-HY1; a polynucleotide comprising the full length protein coding sequence of SEQ ID NOS: 6 or 24 which polynucleotide is assembled from the cDNA insert of clone pEGFR-HY2, the cDNA insert of pEGFR-HY3 and the cDNA insert of clone pEGFR-HY1; or a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NOS: 6 or 24 which polynucleotide is assembled from the cDNA insert of clone pEGFR-HY2, the cDNA insert of clone pEGFR-HY3 and the cDNA insert of clone pEGFR-HY1. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes to the complement of the nucleotide sequence of SEQ ID NOS: 1, 2, 5 or 23 under stringent hybridization conditions; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide of SEQ ID NO: 3, 6, or 24, or amino acids 1-502 of SEQ ID NO: 4. Contemplated allelic variants include those comprising the nucleotide sequences set forth in SEQ ID NO: 27, 29 or 31, the mature protein coding portions thereof, or fragments thereof encompassing the portions that differ in nucleotide sequence compared to SEQ ID NO: 23. Such fragments are particularly useful as probes to identify alleles and include fragments encompassing nucleotides 271 to 288 of SEQ ID NO: 27, nucleotides 271 to 279 of SEQ ID NO: 29, or nucleotides 1440-1442 of SEQ ID NO: 31.
The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NOS: 3, 6 or 24; a polypeptide comprising amino acid residues 1-502 of SEQ ID NO: 4; a polypeptide comprising amino acid residues 1-21 of SEQ ID NOS: 6 or 24; a polypeptide comprising amino acid residues 80-93 of SEQ ID NOS: 6 or 24; a polypeptide comprising amino acid residues 95-128 of SEQ ID NOS: 6 or 24; a polypeptide comprising amino acid residues 133-168 of SEQ ID NOS: 6 or 24; a polypeptide comprising amino acid residues 175-214 of SEQ ID NO:6; a polypeptide comprising amino acid residues 220-259 of SEQ ID NOS: 6 or 24; a polypeptide comprising amino acid residues 446-465 of SEQ ID NOS: 6 or 24; or a polypeptide comprising amino acid residues 363-365 of SEQ ID NOS: 6 or 24. The polypeptide of SEQ ID NOS: 6 or 24 has been designated EGFL6.
The polypeptides of the present invention further include, but are not limited to, a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGFR-HY1 deposited with the ATCC; a polypeptide comprising the amino acid encoded by the cDNA insert of clone pEGFR-HY2 deposited with the ATCC; a polypeptide comprising the amino acid encoded by the cDNA insert of clone pEGFR-HY3 deposited with the ATCC; a full length protein of SEQ ID NO:6 or 24 comprising the amino acid sequence encoded by the cDNA insert of clone pEGFR-HY2, nucleic acids 323-357 of SEQ ID NOS: 5 or 23 and the cDNA insert of clone pEGFR-HY1, or; a mature protein coding sequence of SEQ ID NOS: 6 or 24 comprising the amino acid sequence encoded by the cDNA insert of clone pEGFR-HY2, nucleic acids 323-357 of SEQ ID NOS: 5 or 23 and the cDNA insert of clone pEGFR-HY1. The polypeptides of the present invention also include, but are not limited to, a full length protein of SEQ ID NO:6 or 24 encoded by the open reading frame (ORF) assembled from the cDNA insert of clone pEGFR-HY2, the cDNA insert of clone pEGFR-HY3 and the cDNA insert of clone pEGFR-HY1; or a mature protein coding sequence of SEQ ID NOS: 6 or 24 encoded by the ORF assembled from the cDNA insert of clone pEGFR-HY2, the cDNA insert of clone pEGFR-HY3 and the cDNA insert of clone pEGFR-HY1. Polypeptides of the invention include isoforms encoded by the allelic variants of SEQ ID NOS: 27, 29 or 31, mature protein portions thereof, or fragments of at least about 5 amino acids encompassing the portions that differ in amino acid sequence compared to SEQ ID NO: 24. Polypeptides comprising such fragments may be useful in generating antibodies specific for the isoforms, and include fragments encompassing amino acid 28 to 33 of SEQ ID NO: 28, amino acid 28 to 30 of SEQ ID NO: 30, or amino acid 395 of SEQ ID NO: 32.
Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The invention also relates to methods for producing a polypeptide comprising growing a culture of the cells of the invention in a suitable culture medium, and purifying the protein from the culture. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for stimulation of epithelial tissue growth, e.g., skin regeneration. The polypeptides and polynucleotides of the invention may, therefore, be utilized, for example, as part of methods for tissue repair and regeneration, corneal transplant healing, bum treatment, skin graft production and administration, and wound healing, e.g., treatment of surgical incisions, and ulcers, such as stomach or diabetic ulcers. In addition, the polynucleotides and polypeptides of the invention can further be utilized, for example, as part of methods for the prevention and/or treatment of disorders involving cell fate and differentiation, such as leukemias, brain tumors (including meningiomas, glioblastoma multiforme, anaplastic astrocytomas, cerebellar astrocytomas, other high-grade or low-grade astrocytomas, brain stem gliomas, oligodendrogliomas, mixed gliomas, other gliomas, cerebral neuroblastomas, craniopharyngiomas, diencephalic gliomas, germinomas, medulloblastomas, ependymomas. choroid plexus tumors, pineal parenchymal tumors, gangliogliomas, neuroepithelial tumors, neuronal or mixed neuronal glial tumors), lung tumors (including small cell carcinomas, epidermoid carcinomas, adenocarcinomas, large cell carcinomas, carcinoid tumors, bronchial gland tumors, mesotheliomas, sarcomas or mixed tumors), prostate cancers (including adenocarcinomas, squamous cell carcinoma, transitional cell carcinoma, carcinoma of the prostatic utricle, or carcinosarcomas), breast cancers (including adenocarcinomas or carcinoid tumors), or gastric, intestinal, or colon cancers (including adenocarcinomas, invasive ductal carcinoma, infiltrating or invasive lobular carcinoma, medullary carcinoma, ductal carcinoma in situ, lobular carcinoma in situ, colloid carcinoma or Paget""s disease of the nipple), skin cancer (including melanoma, squamous cell carcinoma, tumor progression of human skin keratinocytes, basal cell carcinoma, hemangiopericytoma and Karposi""s sarcoma), lymphoma (including Hogkin""s disease and non-Hodgkin""s lymphoma), sarcomas (including osteosarcoma, chondrosarcoma and fibrosarcoma) as well as for the treatment of nervous system disorders.
The methods of the present invention further relate to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited above and for the identification of subjects exhibiting a predisposition to such conditions. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
The EGFL6 genes of the present invention is expressed in certain cancer cells, particularly meningiomas, lung tumors, and has been localized to chromosome X, aberrations in which have been implicated in meningiomas and lung tumors. EGFL6 mRNA has also been shown to be differentially expressed in tonsil, placenta, breast carcinomas, prostate carcinomas, lung carcinomas, brain tumors, skin tumors, sarcomas, lymphomas and colon carcinomas while having only low expression in normal breast and normal lung and no detectable expression in normal prostate, lung, brain, skin, skeletal and smooth muscle, lymph nodes or colon. EGF motif-containing molecules have been previously linked to the progression of various cancers. In addition, EGFL6 mRNA expression was detected at all grades and stages of cancer tested indicating EGFL6 expression is detectable at low grades. Highly specific and significant expression of EGFL6 in tumor cells indicates that this protein represents a potential marker of malignancy and a potential candidate for small molecule therapeutic development for the treatment of certain tumors. Expression of EGFL6 has been shown to promote cellular proliferation. Thus, compounds that inhibit the activity of EGFL6 polypeptides, including variants thereof (having preferably at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 24), are expected to reduce undesirable cellular proliferation, particularly cancer cell generation, proliferation or metastasis. Such compounds include antibodies or fragments thereof, antisense polynucleotides, or small molecule modulators of EGFL6 receptor-binding or other activity.
Moreover, the addition of EGFL6 to cell culture or the expression of EGFL6 by cells in culture may enhance proliferation of the cells being cultured, particularly where cells are undifferentiated (e.g. precursor or progenitor cells) or dedifferentiated cells.
Thus, the prognostic and diagnostic methods contemplated according to this aspect of the invention include methods of detecting or quantitating EGFL6 polypeptides in tissue s (e.g., biopsied tissue from brain, lung, breast, prostate, colon, intestine, stomach, or other tissues) or body fluid s (e.g., cerebrospinal fluid, pleural fluid, sputum, ascites, blood, urine, feces, prostatic fluid or other fluids), particularly for diagnosis, prognosis or monitoring of cancer. For these methods of detecting the level of EGFL6 polynucleotide or polypeptide in tissues and bodily fluid, the level of EGFL6 detected is correlated with a standard indicative of the diagnosis of cancer.
The invention provides for methods of detecting cancerous cell expressing the EGFL6 polynucleotide, including but not limited to prostate cancer cells, breast cancer cells, colon cance cells, brain cancer such as meningoma and astrocytoma, skin cancer cells such as melanoma, lymphoma cells and sarcoma cells, comprising the step of contacting the a biological sample with a labeled polynucleotide complementary to an EGFL6 polynucleotide or a fragment thereof for a period sufficient to form a complex; and detecting the complex so that if a complex is detected, the cancerous cell is detected These methods include detecting the EGFL6 polynucleotide comprising SEQ ID NO: 23, a polynucleotide fragment of SEQ ID NO: 23, a nucleotide sequence encoding the mature protein coding portion of SEQ ID NO: 24 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 23. These methods also include detecting a cancerous cell comprising expressing the EGFL6 polypeptide in a biological sample comprising contacting the sample with an antibody or fragment thereof that specifically binds to the EGFL6 polypeptide or a fragment thereof for a period sufficient to form a complex and detecting the complex, so that if a complex is detected, the cancerous cell is detected. The EGFL6 polypeptides include a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a polypeptide fragment of SEQ ID NO: 24, such as a polypeptide fragment comprises amino acids 412 to 424 of SEQ ID NO: 24, polypeptide fragment comprising the mature protein sequence of SEQ ID NO: 24 or a polypeptide with 90% sequence identify to SEQ ID NO: 24. Biological samples include tissue samples and cell samples including those extracted by biopsy or surgery, and biological fluids including but not limited to serum, blood, urine, lymphatic fluid and cerebrospinal fluid. The polypeptide complementary to SEQ ID NO: 24 may be labeled with a radio isotope, affinity label, enzymatic label or a fluorescent label.
The invention also provides methods for the identification of compounds that modulate the expression of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited above. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. For example, assays may include the step of measuring EGFL6-induced cell proliferation in the presence of and absence of a test compound. Candidate inhibitors identified by these methods are also contemplated.
The methods of the invention also include methods for the treatment of disorders as recited above which may involve the administration of such compounds to individuals exhibiting symptoms or tendencies related to disorders as recited above. In addition, the invention encompasses methods for treating diseases or disorders as recited above by administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene expression or target protein activity.
The linkage of EGFL6 with cancer indicates that inhibitors of its activity (that either inhibit expression of the gene product or inhibit activity of the gene product itself) may be useful in treating cancer conditions. Such inhibitors include antisense polynucleotides, antibodies, and other modulators identified through, e.g., screening of libraries or combinatorial libraries of inorganic or organic compounds (such as bacterial, fungal, mammalian, insect or plant products, peptides, peptidomimetics and organomimetics). Such modulators may be administered parenterally, including into the cerebro-spinal fluid, or locally via an implant or device.
The present invention demonstrates that expression of an EGFL6 polypeptide increases tumor cell tumorgenicity in vitro as indicated by colony formation in soft agar (See Example 13). Tumorgenic cells have a transformed phenotype and possess the properties necessary to form tumors. Tumorgenic cells include malignant cells. In tumor progression, the transformed cells proceed through a multi-step process from a transformed phenotype to a neoplastic phenotype and eventually to a metastatic phenotype. The growth characteristics of transformed cells in vitro include immortality, anchorage independence, loss of contact inhibition, reduced density dependence, low serum requirement, growth factor independence, high plating efficiency and shorter population doubling time. The genetic properties of transformed and tumorgenic cells include a high spontaneous mutation rate, anuploidy and heteroploidy. The properties of a neoplastic cell include tumorgenicity, angiogenicity, invasiveness and enhanced protease secretion. Tumorgenicity can be assessed by assays well known in the art which measure any of the properties listed above.
The methods of the invention include methods of reducing tumor size in a patient suffering from cancer, including but not limited to prostate cancer, breast cancer, colon cancer, brain cancer such as meningoma and astrocytoma, and skin cancer such as melanoma, lymphoma and sarcoma, comprising administering to the patient an inhibitor of EGFL6 activity wherein the inhibitor binds the EGFL6 polypeptide, such as the polypeptide comprising the amino acid sequence of SEQ ID NO: 24, the mature protein sequence of SEQ ID NO: 24 or a EGFL6 receptor polypeptide. Inhibitors of EGFL6 activity include antibodies and fragments thereof, peptides, and small molecules. The inhibitor may also be an antisense polynucleotide which binds to the polynucleotide, or a fragment or variant thereof, which encodes the mature protein coding portion of SEQ ID NO: 24.
The present invention also provides for methods of inhibiting tumorgenicity in a cell expressing an EGFL6 polypeptide comprising the step of contacting said cells with an antibody or fragment thereof that specifically binds the polypeptide of SEQ ID NO: 24 or a fragment thereof, or contacting said cell with an antisense polynucleotide that specifically binds a polynucleotide, or a fragment or variant thereof encoding the mature protein coding portion of the polypeptide of SEQ ID NO: 24. These methods include contacting cells which are present in a subject suffering from cancer, including but not limited to prostate cancer, breast cancer, colon cancer, brain cancer such as meningoma and astrocytoma, and skin cancer such as melanoma, lymphoma and sarcoma.
The invention provides for methods of inhibiting proliferation of cancer cells, comprising the step of contacting the cells with an antibody or fragment thereof that specifically binds the polypeptide of SEQ ID NO: 24 or a fragment thereof, or contacting said cell with an antisense polynucleotide that specifically binds a polynucleotide, or a fragment or variant thereof which encodes the mature protein coding portion of the polypeptide of SEQ ID NO: 24. These methods include contacting cells which are present in a subject suffering from cancer, including but not limited to prostate cancer, breast cancer, colon cancer, brain cancer such as meningoma and astrocytoma, and skin cancer such as melanoma, lymphoma and sarcoma.
The invention provides for pharmaceutical compositions comprising an amount of an antibody or fragment thereof that specifically binds to a polypeptide of SEQ ID NO: 24 in a pharmaceutically acceptable carrier, wherein the amount of antibody or fragment thereof effectively inhibits EGFL6 polypeptide activity, such as those activities described herein, reduces tumor size, inhibits cancer cell proliferation or inhibits tumorgenicity. The invention also provides for pharmaceutical compositions comprising an amount of an antisense polynucleotide that specifically binds to a polynucleotide encoding the mature protein coding portion of SEQ ID NO: 24 in a pharmaceutically acceptable carrier, wherein the amount of antisense polynucleotide effectively inhibits EGFL6 polypeptide activity, such as those activities described herein, reduces tumor size, inhibits cancer cell proliferation or inhibits tumorgenicity